The long term objective of this Revised Competing Continuation Grant Proposal is to define the regulatory systems affecting the function of Na+:K+(NH4+):2Cl- cotransporters and K+(NH4+) channels located in apical membranes of thick ascending limb of Henle [MTAL] cells isolated from the inner stripe of the outer medulla of the mammalian kidney. The importance of these studies is based on the hypothesis that active transport of K+(NH4+) by the MTAL provides the critical "single effect" for countercurrent multiplication processes which generate high concentrations of K+ and NH4+ in the renal medulla. The accumulation of K+ and NH4+ in the renal medulla, in turn, regulates the excretion of these ions by the terminal segments of the kidney nephron. Thus identification and characterization of the hormones/factors and their associated second messenger systems that regulate these transporters is required to achieve an in-depth understanding of renal K+ and NH4+ (or acid) excretion. Disorders affecting these processes could profoundly alter body potassium and acid-base balances. This proposed research project will utilize three cell systems from the mouse kidney: isolated perfused single MTAL tubules, suspensions of MTAL tubules/cells obtained by immunoisolation, and primary cultures of MTAL cells. Quantitative electrophysiological, morphological, fluorescence, biochemical and isotopic flux approaches will be employed: (1) to assess the role of arginine vasopressin (AVP)-V1 and -V2 receptor agonists [and other hormones/factors known to modulate cotransporter activity] and their associated second messenger systems in modulating the apical cotransporter between Na+:Cl- and Na+:K+(NH4+):2Cl- cotransport modes and in modulating the activity of K+(NH4+) channels; (2) to determine the effect of cell pH in altering the K+(NH4+) transport mode of the apical Na+:Cl- cotransporter; and (3) to evaluate the role of AVP-V1 and -V2 receptor agonists and their associated second messenger systems in modulating cell pH via their effects on known acid-base transporters [in particular, the apical and basolateral Na+:H+ antiporters].